Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential (ΔΨ_m), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co ²⁺ (CC) staining can be dissociated from the ΔΨ _m loss, both of which may occur in a caspasedependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent ΔΨ_m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.